Diabetes mellitus is the most prevalent metabolic disease of humans. The most common form of diabetes is associated with peripheral insulin resistance, a condition whereby muscle tissue fails to respond to elevated circulating insulin with the appropriate uptake and disposal of blood glucose in the postprandial state. In a previous grant period we identified and cloned Glut4, the facilitative glucose transporter isoform that is the key rate-limiting component in insulin-stimulated skeletal muscle glucose uptake. Current evidence suggests that insulin resistance results from a disruption in the normal mechanism by which insulin activates Glut4 in skeletal muscle. The long-term goal of this proposal is to elucidate the molecular mechanism of insulin-stimulated glucose transport and to identify the defects in this process that predispose to diabetes mellitus. To accomplish this goal, the following specific aims will be undertaken in the next project period: l. Insulin stimulates glucose transport in fat and muscle by altering the subcellular trafficking of Glut4 and causing its redistribution from an intracellular storage compartment to the plasma membrane. We have identified a region within the C-terminal cytoplasmic tail of Glut4 that confers insulin-regulated subcellular targeting. Site-directed mutagenesis will be used in conjunction with an insulin-responsive L6 myocyte expression-trafficking system to completely define the role of the dileucine targeting motif and the adjacent serine phosphorylation site in the subcellular tracking of Glut4 in a native, insulin-sensitive cell type. 2. Little is known about the cellular machinery involved in the subcellular trafficking and regulation of Glut4. The yeast 2-hybrid cloning system will be used to identify and clone novel proteins that interact directly with the known C-terminal targeting domain of Glut4. 3. We have generated and characterized transgenic mice overexpressing Glut1 or Glut4 in skeletal muscle. These mice exhibit enhanced muscle glucose uptake and disposal and have dramatically improved glucose tolerance relative to control littermates. The ability of the glucose transporter transgenes to confer resistance to hyperglycemia will be evaluated in several experimental models of diabetes, thus forming the basis for a potential gene therapy strategy for diabetes.